CN1981187A - Portable sample analyzer - Google Patents

Abstract

A sample analyzer cartridge for use at a point of care of a patient such as in a doctor's office, in the home, or elsewhere in the field. By providing a removable and/or disposable cartridge with all of the needed reagents and/or fluids, the sample analyzer can be reliably used outside of the laboratory environment, with little or no specialized training.

Description

Portable Sample Analyzer Box

This application claims priority to US Patent Application Serial No. 60/571,235, filed May 14, 2004, which is hereby incorporated by reference.

Background technique

The present invention relates generally to sample analyzers and, more particularly, to sample analyzers and cartridges that may be used at a patient's point of care, such as a doctor's office, home, or other site.

Particle discrimination can be used in a variety of clinical assays such as determining cell number and type in blood samples, detecting bacterial or viral particles in body fluid samples and assaying cell volume and concentration. Detection of non-cellular particles such as proteins and analytes in urine samples is also of value in certain clinical tests, for example. Analysis of crystals and other particulates in fluid suspensions also has important industrial uses.

A method that can quickly and efficiently distinguish particles in a particle suspension sample is flow cytometry. In this method, a suspension of particles in a blood sample (typically cells in the blood sample) is transported through a fluid channel, wherein individual particles in the sample are illuminated with one or more focused light beams. Interaction of the light beam with individual particles flowing through the fluidic channel is detected by one or more photodetectors. Typically, these detectors are designed to measure absorption or fluorescence emission at specific beam or emission wavelengths and/or light scattering at specific scattering angles. Thus, each particle flowing through a fluidic channel can be characterized by one or more properties related to its absorption, fluorescence, light scattering, or other optical or electrical properties. The properties measured by these detectors allow each particle to be mapped to a certain characteristic space, where the axis of this space is the light intensity or other properties measured by the detectors. Ideally, the different particles in the sample are mapped to distinct and non-overlapping regions of the feature space, so that each particle can be analyzed based on the mapping of these particles in the feature space. Such analysis may include counting, identifying, quantifying (with respect to one or more physical characteristics), and/or classifying the particles.

In flow cytometry, two types of light scatter measurements are routinely performed. Light intensity measurements at small angles (approximately 1.5-13 degrees relative to the incident beam) are often called forward or small angle scatter and provide information about cell size. To a large extent, forward scatter also correlates with the difference in refraction between cells and the extracellular medium, allowing the identification of cells with damaged membranes. Measurements of light intensity at angles of about 65°-115° relative to the incident light, often referred to as orthogonal or large-angle scatter, can provide information about the size and degree of structure of the particles.

Simultaneous light scattering measurements at different angles or combining such light scattering measurements with absorption or fluorescence measurements have been proposed in flow cytometry. For example, light absorption combined with light scattering can be used in flow cytometry to distinguish red blood cells from platelets, and to distinguish lymphocytes, monocytes, basophils, eosinophils, and neutrophils. However, this method sometimes requires staining of the cells, so it is quite complicated and it may not be possible to use the cells for subsequent studies after sorting the cells.

Light scattering measurements combining circular dichroism (CD) and optical dispersion (ORD) can also be used for particle differentiation in suspensions of viral particles or cells. Studies on the effect of Mie (isotropic particle) scattering on the CD and ORD spectra of DNA in virus particles have shown that scattering measurements can be used to correct ORD and CD measurements in larger biological structures such as virus particles and cells Values to allow particle differentiation based on ORD and CD properties. Differential scattering of right- and left-circularly polarized light for differentiating between several different microorganisms is also demonstrated. The circular intensity differential scattering (CIDS) method is similar to CD (using differential absorption of left and right circularly polarized light), but the CIDS method uses a helical structure (such as DNA) to differentially scatter right and left circularly polarized light.

Typically, such particle differentiation is performed at least in part using one or more devices, collectively referred to herein as a sample analyzer. Many sample analyzers are fairly large devices used by trained personnel in a laboratory setting. In order to use many sample analyzers, the collected sample must first be processed (such as diluting the sample to the desired level, adding appropriate reagents, and centrifuging the sample to provide the desired separation) before the prepared sample can be provided to the sample analyzer. things, etc.). To achieve accurate results, this type of sample handling typically must be performed by trained personnel, which can add to the cost and time required to perform sample analysis.

During the analysis phase, many sample analyzers also require operator intervention (eg, requiring additional information entry or other processing of the sample). This may further increase the cost and time required to perform the desired sample analysis. Furthermore, many sample analyzers output only raw analytical data, often requiring further calculations and/or interpretation by trained personnel in order to make appropriate clinical decisions.

Contents of the invention

The present invention relates generally to sample analyzers and, more particularly, to sample analyzers with removable and/or disposable cartridges for use at a patient's point of care, such as a doctor's office, home, or other site. By providing removable and/or disposable cartridges with all necessary reagents and/or fluids, the sample analyzer can be reliably used outside of a laboratory environment with little or no specialized training required. This can help streamline the sample analysis process, reduce the cost and workload of medical staff or others, and, for many patients (including those who require relatively frequent blood monitoring/analysis), increase the throughput of sample analysis. convenience.

In one illustrative embodiment of the invention, a sample analyzer is provided that has a removable cartridge that can receive a collected sample, such as a collected whole blood sample, and, once the removable cartridge is installed and activated With the analyzer installed, these analyzers and cartridges automatically process the sample, and the analyzer provides the user with sufficient information to make a clinical decision. In certain embodiments, the analyzer displays or prints quantitative results (eg, within and/or outside of predefined ranges), which eliminates the need for further calculations or interpretation by the user.

For example, sample analyzers may be used to determine the number and/or type of cells in a blood sample, detect bacterial or viral particles in fluid, food or other samples, and/or cell volume and density in assay samples. The sample analyzer may also be used to detect non-cellular particulates, such as uric acid crystals in urine samples and crystals and other particulates in fluid suspensions, and/or for any other suitable type of analysis as desired.

In one illustrative embodiment, an analyzer includes a housing and a removable fluid cartridge, wherein the housing is adapted to receive the removable fluid cartridge. In some cases, the removable fluid cartridge is a disposable cartridge. In an illustrative embodiment, a removable fluid cartridge may contain one or more reagents (e.g., solvents, spheronizing reagents, diluents, etc.), one or more analytical channels, one or more flow sensors, one or more valves and/or fluidic circuits suitable for processing (eg, lysing, spheroidizing, diluting, mixing, etc.) the sample and sending the processed sample to the appropriate analysis channel on the cartridge. To support the card, the housing may include a pressure source, one or more light sources, one or more light detectors, a processor and a power supply. A pressure source can provide suitable pressure to the removable fluid cartridge ports to allow fluid flow in the fluid circuit as desired. The prepared sample in at least selected analysis channels of the removable cartridge can be interrogated with the one or more light sources of the analyzer, and the one or more light detectors of the analyzer can detect passing, sampled samples Absorbed and/or scattered light. A processor can be coupled to at least some of the light sources and detectors, and can determine one or more parameters of the sample. In some embodiments, the above-mentioned one or more analysis channels on the detachable fluid cartridge may include one or more flow cytometry channels and/or one or more absorption channels, but not required to have in all embodiments this configuration.

In certain demonstrative embodiments, a whole blood sample may be provided to a removable fluid cartridge, and the removable cartridge is adapted to perform blood analysis, such as a complete blood count (CBC) analysis. To count and classify red blood cells in a whole blood sample, a portion of the whole blood sample can be separated and provided to the red blood cell measurement channel of the removable cartridge. If desired, the portion of the blood sample provided to the RBC measurement channel can be diluted, the RBCs can be spheroidized in real time, the resulting sample can be hydrodynamically concentrated to form nuclei, and the resulting nuclei can be provided in a removable cartridge The first flow cell channel.

In some cases, a first flow cytometric channel can be positioned along or below a first transparent fluid window in a removable cartridge such that a first light source and a first light detector can optically interrogate cells/ platelets. A flow sensor may also be provided on the removable cartridge to measure the flow rate through the first flow cell channel.

In some cases, measured parameters may include, sample flow rate (FR), measurement time (T) length, sample dilution factor (DF), counted red blood cell number (N _RB ), counted platelet number (N _Plt ), The diameter (drbc) of each cell and the hemoglobin concentration (CHC) of each cell. Several red blood cell analysis parameters can be calculated from these parameters, including red blood cell count (RBC), platelet count (Plt), mean cell hemoglobin concentration (MCHC), mean cell volume (MCV), mean cell hemoglobin content (MCH), relative distribution width ( RDW), hematocrit parameters (Hct) and/or hemoglobin concentration (Hb).

In certain embodiments, a portion of the blood sample may also be directed to the absorption measurement channel on the removable cartridge. The absorption measurement channel can be arranged along the transparent window so that the corresponding light source and light detector can optically interrogate the blood sample. If desired, another flow sensor can be provided on the removable cartridge to measure the flow rate into or through the absorption measurement channel, but this is not required. Absorption channels can be used to measure the level of absorption of incident light. This level of absorption can provide a measure of the average or total cellular hemoglobin concentration in the blood sample.

For counting and classifying white blood cells, a portion of the whole blood sample can be separated and provided to the white blood cell measurement channel in the removable cartridge. If desired, the portion of the blood sample provided to the leukocyte measurement channel can be diluted, the red blood cells therein can be lysed instantaneously, and the resulting sample hydrodynamically focused to form nuclei that are finally provided to the Second flow cell channel in removable cartridge. A second flow cell channel may also be positioned along or below the transparent fluid window in the removable cartridge, allowing corresponding light sources and detectors to optically interrogate the cells in the fluid stream. In some cases, a flow sensor can be provided on the removable cartridge to measure the flow rate through the second flow cytometric channel.

In some cases, illustrative measurement parameters of a WBC measurement channel may include three (3) or (5) partial WBC differentials, total WBC count, and/or on-axis WBC volume. Depending on the desired application, other parameters may also be measured or calculated. In some cases, staining and/or fluorescent labels can be added to the sample prior to providing the sample to the second flow cytometry channel, which in some cases facilitates cell differentiation.

Description of drawings

Figure 1 is a perspective view of a sample analyzer and cartridge according to the present invention;

Figure 2 is a schematic diagram of the sample analyzer and cartridge of Figure 1;

Figure 3 is a more detailed schematic diagram showing the flow control of the sample analyzer and cartridge in Figure 2;

Figure 4 is a schematic illustration of certain features of a cartridge according to the invention;

Figure 5 is a schematic diagram of several storage pools that may be included in a cartridge according to the invention;

Figure 6 is a flowchart illustrating a method of analyzing a blood sample according to the present invention;

Figure 7 is a flowchart illustrating a method of obtaining several red blood cell parameters according to the present invention;

Figure 8 is a flowchart showing another method of analyzing a blood sample according to the present invention;

Figure 9 is a flowchart illustrating a method of operating a sample analyzer in accordance with the present invention;

Figure 10 is a flowchart illustrating another method of operating a sample analyzer in accordance with the present invention;

Figure 11 is a flowchart illustrating another method of operating a sample analyzer in accordance with the present invention; and

Figure 12 is a flowchart showing another method of operating a sample analyzer in accordance with the present invention.

Detailed ways

Figure 1 is a perspective view of a sample analyzer and cartridge according to the present invention. In the Figures, the sample analyzer is indicated at 10 and includes a housing 12 and a removable or disposable cartridge 14 . The housing 12 includes a base 16, a cover 18, and a hinge 20 connecting the base 16 to the cover 18, but this is not required. In this embodiment, the base 16 includes a first light source 22a, a second light source 22b, and a third light source 22c, as well as associated optics and necessary electronics for operating the sample analyzer. Depending on the application, each light source can be a single light source or consist of multiple light sources. In some embodiments, the overall size of the housing may be less than 1 cubic foot, less than one-half cubic foot, less than one-quarter cubic foot, or less, as desired. Similarly, the total weight of the housing can be less than 10 pounds, less than 5 pounds, less than 1 pound, or less, as desired.

Cover 12 includes a pressure source, such as a pressure chamber with microvalve control, a first photodetector 24a, a second photodetector 22b, and a third photodetector 22c, and these elements have associated optics and electronics . Depending on the application, each photodetector can be a single detector or consist of multiple detectors. Depending on the application, polarizers and/or filters may also be provided as required.

Removable cartridge 14 is adapted to receive sample fluid through a sample collector port, which in this embodiment includes a lancet 32 . In some embodiments, the lancet 32 is retractable and/or spring loaded. When removable cartridge 14 is not in use, sheath 38 may be used to protect sample collector port and/or lancet 32 .

In this embodiment, the removable cartridge 14 performs blood analysis on a whole blood sample. A user's finger can be pricked with a lancet 32 to produce a blood sample that can be drawn by capillary action into an anticoagulant-coated capillary in the removable cartridge 14 . A removable cartridge 14 similar to the fluid circuits offered by Micronics Technologies, Inc., some of which are fabricated from a layered structure with etched channels, can be fabricated. However, it is believed that the removable case 14 may be manufactured in any suitable manner, including by injection molding or any other suitable manufacturing process and method, as desired.

During use and after a blood sample has been drawn into the removable cartridge 14, the removable cartridge 14 can be inserted into the housing with the lid 18 in the open position. In some cases, removable case 14 may include holes 26a and 26b for receiving alignment pins 28a and 28b in base 16, which facilitate alignment and connection between the different parts of the instrument. The removable box 14 may also include a first transparent fluid window 30a, a second transparent fluid window 30b and a third transparent fluid window 30c, which are respectively connected to the first, second and third light sources 22a, 22b and 22c, and the first, second and third light sources 22a, 22b and 22c, The second and third photodetectors 24a, 24b and 24c are aligned.

In removable cartridge 14, when the cover is moved to the closed position and the system is pressurized, cover 18 may provide controlled pressure to pressure receiving ports 34a, 34b, 34c via pressure providing ports 36a, 36b, 36c, and 36d and 34d. We believe that, depending on the application, more or fewer pressure supply and pressure receive ports can be used. Alternatively or additionally, it is contemplated that one or more micropumps (such as electrostatically actuated midi pumps) could be placed on or in the removable cartridge 14 to provide the necessary pressure to operate the fluid on the removable cartridge 14 circuit. Certain exemplary electrostatically actuated midsize pumps are described in U.S. Patent Nos. 5,836,750, 6,106,245, 6,179,586, 6,729,856, and 6,767,190, all of which are assigned to the assignee of the present invention, which are incorporated herein by reference These patents are incorporated herein.

Once pressurized, the shown instrument performs blood analysis on the collected blood sample. In some cases, blood analysis may include complete blood count (CBC) analysis, however, depending on the application, other types of analysis may also be performed.

For counting and sorting red blood cells, a portion of the whole blood sample can be separated and provided to the red blood cell measurement channel in the removable cartridge 14 . If desired, the blood sample can be diluted, the red blood cells therein can be spheroidized in real time, and the resulting sample hydrodynamically concentrated to form nuclei, which are finally provided to the first flow cell channel. A first flow cell channel may be positioned along a first transparent fluid window 30a of the removable cartridge 14, which allows the first light source 22a and first light detector 24a to optically interrogate cells in the fluid stream. In some cases, a first flow sensor may be provided on the removable cartridge 14 to measure the flow rate through the first flow cell channel.

In some cases, measured parameters may include, sample flow rate (FR), measurement time (T) length, sample dilution factor (DF), counted red blood cell number (N _RB ), counted platelet number (N _Plt ), Diameter of individual cells (drbc) and hemoglobin concentration (CHC) of individual cells. From these parameters, several red blood cell analysis parameters can be calculated, including red blood cell count (RBC = N _RB / (DF × FR × T)), platelet count (Plt = N _plt / (DF × FR × T)), mean cell hemoglobin concentration (MCHC=<CHC>), mean cell volume (MCV=(π/6)×<drbc ³ >), mean cell hemoglobin content (MCH=(π/6)×<drbc ³ ×CHC>), relative distribution width (RDW=[(π/6)×drbc ³ ]/standard deviation of MCV), hematocrit parameters (Hct=RBC×MCV) and/or hemoglobin concentration (Hb=MCHC×Hct).

In some embodiments, a portion of the blood sample may also be introduced into the absorption measurement channel. An absorption measurement channel may be provided along the third transparent window 30c of the removable cartridge 14, which allows the third light source 22c and third light detector 24c to optically interrogate the blood sample. A flow sensor may be provided on the removable cartridge 14 to measure the flow rate through the absorption measurement channel. The absorption measurement channel may measure the absorption of incident light provided by the third light source 22c. The measured absorption level can provide an indication of the total or average cellular hemoglobin concentration in the blood sample.

For counting and classifying white blood cells, a portion of the whole blood sample can be separated and provided to the white blood cell measurement channel in the removable box 14 . If desired, the blood sample can be diluted, red blood cells can be lysed in real time, and the resulting sample can be hydrodynamically concentrated to form nuclei, which are then provided to a second flow cell channel. A second flow cell channel may be positioned along the second transparent fluid window 30b of the removable cartridge 14, which allows the second light source 22b and second light detector 24b to optically interrogate the cells in the fluid flow. A flow sensor is provided on the detachable box 14 to measure the flow velocity through the channel of the second flow cytometer. In some instances, measured white blood cell parameters may include, three (3) or (5) fractional white blood cell differentials, total white blood cell count, and/or on-axis white blood cell volume. Depending on the application, other parameters may also be measured or calculated.

Figure 1 shows a sample analyzer and cartridge assembly. However, we believe that other sample analyzer configurations may also be used. For example, the sample analyzer 10 and removable cartridge may be similar to the devices described in Schwichtenberg et al., US Patent Application 2004/0211077, which is incorporated herein by reference.

In some cases, sample analyzer 10 is adapted for use at the point of care of a patient, such as a doctor's office, home, or other site. Providing a sample analyzer 10 that can be reliably used outside of a laboratory environment (requiring little or no specialized training) helps to streamline the sample analysis process, reduces cost and workload for medical staff, and is useful for many patients, including those requiring For those who do relatively frequent blood monitoring/analysis), the convenience of sample analysis is increased.

In operation, sample analyzer 10 can receive a collected sample, such as a collected whole blood sample, and, once the analyzer is activated, sample analyzer 10 can automatically process the sample and provide information to the user to make a clinical decision. In some embodiments, sample analyzer 10 may display or print out quantitative results (eg, within and/or outside predefined ranges) so that no additional calculations or interpretations are required by the user.

FIG. 2 is a schematic diagram of the sample analyzer and cartridge of FIG. 1 . As noted above, in the illustrated embodiment, the base 16 includes a number of light sources 22, associated optics and necessary control and processing electronics 40 for operating the analyzer. The base 16 may also include a battery 42, transformer or other power source. In the figure, the cover 12 is shown with a pressure source/flow control module 44 and several light detectors 24 with associated optical elements.

Removable cartridge 14 may receive sample fluid through a sample collector port or lancet 32 . When pressurized by the pressure source/flow control module 44, the removable cartridge 14 may perform blood analysis on the received blood sample. In certain embodiments, as described above, the removable cartridge 14 may contain various reagents 49 and a fluid circuit for mixing the reagents with the blood sample to prepare the blood sample for analysis. The removable cartridge 14 may also include several flow sensors to help control and/or verify proper operation of the fluid circuit.

In some cases, blood samples are prepared (e.g., lysed, spheroidized, stained, diluted, and/or otherwise processed) and then hydrodynamically pooled for flow cytometry channels on one or more plates Nuclei are formed in (eg flow cell channel 50). In this embodiment, the flow cell channel 50 passes through a transparent fluid window (such as the first transparent fluid window 30 a ) in the detachable cartridge 14 . An array of light sources 22 and their associated optics in base 16 can provide light that passes through the nucleus through fluid window 30a. Likewise, the array of light source 24 and its associated optical elements can receive diffuse and non-scattered light from the nucleus through fluid window 30a. A controller or processor 40 may receive output signals from the array of detectors 24 and may differentiate and/or count selected cells present in the nuclear flow.

It is contemplated that the removable cartridge 14 may also include a fluid control module 48 for helping to control the velocity of at least some of the fluid on the removable cartridge 14 . In the illustrated embodiment, the fluid control module 48 may include flow sensors for sensing the velocity of the various fluids and reporting these velocity to the controller or processor 40 . Controller or processor 40 may then adjust one or more control signals (provided to pressure source/flow control module 44 ) to achieve the desired pressure (and thus the desired fluid velocity) for proper operation of the analyzer.

Because blood and other biological waste fluids may transmit disease, the removable cartridge 14 may include a waste fluid reservoir 52 downstream of the flow cell channel 50 as shown. Waste reservoir 52 may receive and store fluid from removable cartridge 14 . When testing is complete, the removable cartridge 14 can be removed from the analyzer and preferably disposed of in a biological waste compatible container.

FIG. 3 is a more detailed schematic showing the flow control of the sample analyzer and cartridge in FIG. 2 . In this embodiment, pressure source/flow controller 44 in cap 18 provides five controlled pressures, including sample push (P) pressure 36a, solvent (L) pressure 36b, spheroidizer (SP) pressure 36c, sheath Liquid (SH) pressure 36d and diluent (D) pressure 36e. These pressures are illustrative only, and it is believed that more, less or different pressures (eg, dye pressure to the dye reservoir) may be provided by the pressure source/flow controller 44 depending on the application. Likewise, it is contemplated that cover 18 may not include pressure source/flow controller 44 at all. Instead, removable cassette 14 may contain an on-board pressure source (such as a compressed air reservoir), one or more micropumps (such as the electrostatically actuated midi-pump described above), or any other suitable pressure source, as desired. The array consisting of light sources and detectors is not shown in FIG. 3 .

In the illustrated embodiment, pressure source 36a provides pressure to blood sample pool 62 through pusher fluid (pusher fluid) 65, pressure source 36b provides pressure to solvent pool 64, pressure source 36c provides pressure to spheroidizing reagent pool 66, Pressure source 36d provides pressure to sheath fluid reservoir 68 and pressure source 36e provides pressure to diluent reservoir 70 .

In one embodiment, each pressure source may include a first pressure chamber for receiving an input pressure and a second pressure chamber for providing a controlled pressure to the removable cartridge. A first valve may be provided between the first pressure chamber and the second pressure chamber to controllably release pressure in the first pressure chamber to the second pressure chamber. A second valve in fluid communication with the second pressure chamber can controllably vent the pressure in the second pressure chamber to atmosphere. This allows the pressure source/flow controller 44 to provide controlled pressure to each pressure receiving port on the removable cartridge 14 . Each of the The valves may be arrays of individually addressable and controllable electrostatically actuated microvalves, which are incorporated herein by reference. Alternatively, each valve may be an array of electrostatically actuated microvalves that can be pulse-width modulated with a controlled duty cycle to achieve a controlled "effective" flow or leak rate ( leak rate). Other valves can also be used if desired.

The illustrated removable cartridge 14 includes five pressure receiving ports 34 a , 34 b , 34 c , 34 d and 34 e each for receiving a respective controlled pressure from a pressure source/flow controller 44 . In the illustrated embodiment, pressure receiving ports 34a, 34b, 34c, 34d, and 34e introduce controlled pressures into blood pool 62, solvent pool 64, pelleting reagent pool 66, sheath fluid pool 68, and diluent pool 70, respectively. Solvent reservoir 64, pelleting reagent reservoir 66, sheath fluid reservoir 68 and diluent reservoir 70 can be filled prior to delivery of removable cartridge 14, while blood reservoir 62 is filled in the field via sample collector port or lancet 32 .

Flow sensors can be positioned in-line with each or selected fluids as shown. Each flow sensor 80a-80e may measure a corresponding fluid velocity. Preferably, the flow sensors 80a-80e are thermal anemometer type flow sensors, more preferably microbridge type flow sensors. Microbridge flow sensors are described in U.S. Patent Nos. 4,478,076, 4,478,077, 4,501,144, 4,651,564, 4,683,159, and 5,050,429, all of which are incorporated herein by reference. In this article. Output signals from the various flow sensors 80a-80e may be provided to a controller or processor 40. As shown, a controller or processor 40 may provide control signals to a pressure source/controller 44 . For example, to control the pressure provided to the blood sample, the controller or processor 40 may open the first and second pressure chambers in the pressure source/controller 44 when the velocity of the blood sample drops below a first predetermined value. between the first valve to controllably release the pressure in the first pressure chamber to the second pressure chamber. Likewise, when the velocity of the blood sample increases above a second predetermined value, the controller or processor 40 may open the second valve to vent the pressure in the second pressure chamber.

A controller or processor 40 can control the rates of solvent, spheronizing reagent, sheath fluid, and diluent in a similar manner.

In some cases, the controller or processor 40 may detect one or more changes in the flow rate through the fluid channel. Changes in flow rate may correspond to one or more air bubbles, blockage or partial blockage of the fluid channel (caused by coagulation of the blood sample, undesired or foreign objects in the fluid, and/or other undesired properties of the fluid channel). The controller or processor 40 can be programmed to detect such characteristics based on the flow rate and, in some cases, to sound an alarm and/or shut down the sample analyzer.

Thermal anemometer type flow sensors typically include a heater element that, when energized, produces one or more heat pulses in the fluid, and such flow sensors also include an and/or one or more thermal sensors downstream for detecting said one or more thermal pulses. The velocity of the fluid through the fluid channel may be correlated to the time it takes for a heat pulse to travel from the heater component to one of the spaced apart thermal sensors.

In some cases, thermal anemometer-type flow sensors can be used to detect the thermal conductivity and/or specific heat of a fluid. Changes in the thermal conductivity and/or specific heat of the fluid may correspond to changes in the properties of the fluid (such as changes in the state of the fluid (coagulation of blood samples), air bubbles in the fluid, unwanted or foreign objects in the fluid, etc. ). Thus, in some embodiments, it is contemplated that the controller or processor 40 may detect properties of the fluid by monitoring the thermal conductivity and/or specific heat of the fluid passing through a flow sensor of the thermal anemometer type.

In some cases, an impedance sensor may be placed in fluid communication with the fluid channel. A controller or processor 40 may be coupled to the impedance sensor. Changes in fluid impedance may indicate changes in fluid properties (eg, changes in fluid state (coagulation of blood sample), air bubbles in the fluid, unwanted or foreign objects in the fluid, etc.). Thus, in some embodiments, it is contemplated that the controller or processor 40 may detect a property of the fluid by monitoring the impedance of the fluid passing through the impedance sensor.

A downstream valve, indicated generally at 110, may also be provided. The controller or processor 40 can open/close the downstream valve 110 as desired. For example, downstream valve 110 may remain closed until the system is fully pressurized. This helps prevent blood, solvents, spheronizing reagents, sheath fluid, and diluents from flowing into fluid circuit 86 until the system is fully pressurized. Downstream valve 110 may also be controlled to assist in performing certain tests, such as zero flow tests, and the like. In another embodiment, the downstream valve 110 may be opened by mechanical operation (eg, when the lid is closed).

Figure 4 is a schematic illustration of certain features of a removable cartridge according to the present invention. The removable case is indicated generally at 100 and may be similar to the removable case 14 described and shown above in connection with FIGS. 1-3 . It should be understood that removable cartridge 100 is merely illustrative and that the present invention is applicable to many microfluidic cartridges regardless of form, function, or configuration. For example, the present invention can be used in removable cartridges suitable for the following purposes: flow cytometry, hematology, clinical chemistry, blood chemistry analysis, urinalysis, blood gas analysis, virus analysis, bacteria analysis, electrolyte measurement, and the like. We also believe that the removable case of the present invention, such as the removable case 100, may be made of any suitable material or material system, including glass, silicon, one or more polymers, or any other suitable material or material system or multilayer material. A combination of materials or material systems).

The removable cartridge 100 includes a first measurement channel 102 and a second measurement channel 104, although more or fewer measurement channels may be used as desired. In the illustrated embodiment, the first measurement channel 102 is a red blood cell measurement channel and the second measurement channel 104 is a white blood cell measurement channel. Removable cartridge 100 receives a whole blood sample via blood receiving port 106 (by capillary action, drawing a known amount of blood into blood sample storage capillary 108 coated with anticoagulant). A sample pushing (P) pressure (such as sample pushing (P) pressure 36 a of FIG. 3 ) is provided to a sample pushing fluid pool (such as sample pushing fluid pool 65 of FIG. 3 ). When pressure is applied, the sample pushing fluid is forced from the sample pushing fluid pool into the blood sample pushing channel 110 .

In some embodiments, valve 112 and flow sensor 114 may be arranged in-line with blood sample push channel 110 . Valve 112 may be controlled such that it is opened when it is desired to push a blood sample through the fluid circuit. The flow sensor 114 may measure the flow rate of the blood sample push fluid, and thereby the flow rate of the blood sample through the anticoagulant-coated capillary 108 . The flow rate provided by the flow sensor 114 can be used to help control the sample push (P) pressure provided to the removable cartridge 100 .

In the illustrated embodiment, a whole blood sample is split and the split is provided to red blood cell measurement channel 102 and white blood cell measurement channel 104 via branch 116 . In the illustrated embodiment, valve 118 is provided in-line with the aforementioned branches to control the flow of blood sample into the red blood cell measurement channel 102 and valve 120 is provided to control the flow of blood into the white blood cell measurement channel 104. sample stream.

Turning now to the red blood cell measurement channel 102, the red blood cell spheroidizer pressure (SP) (eg, spheroidizer pressure (SP) 36c of FIG. 3) is provided to the spheroidizer pool (eg, spheroidizer pool 66 of FIG. 3). When pressure is applied, the nodulizing reagent in the nodulizing reagent pool 66 is forced into the nodulizing reagent channel 124 .

In some embodiments, valve 126 and flow sensor 128 may also be positioned in-line with spheroidizing reagent passage 124 . Valve 126 can be controlled such that it is opened when it is desired to push the spheronizing agent into the fluid circuit. The flow sensor 128 can measure the flow rate of the nodulizing reagent and measure the flow rate of the nodulizing reagent flowing through the nodulizing reagent channel 124 . The flow rate provided by flow sensor 128 may be used to help control the spheronizing reagent pressure (SP) provided by pressure source/controller 44 to removable cartridge 100 .

During normal functional operation of the removable cartridge 100, the spheroidizing reagent is pushed into the confluence region 130 at the spheroidizing reagent flow rate, and the blood sample is pushed into the confluence region 130 at the blood sample flow rate. The blood sample flow rate and the spheroidizing reagent flow rate can be controlled by the pressure source/controller 44 of FIG. 3 .

The confluence region 130 may be configured such that when the two fluids described above flow through the confluence region 130, the spheroidizing reagent flows around the blood sample. In some cases, the flow rate of the spheroidized reagent can be higher than the blood sample flow rate, which can help improve the flow characteristics in the downstream instant spherized channel 132, and in some cases, help to form a complete and stable flow rate of the spheronized reagent. Thin band of blood evenly surrounded. When the red blood cells flow through the instant spheroidization channel 132, this ribbon flow can help the spheroidizing agent to evenly terraform the red blood cells. Furthermore, the length of the instant spheroidizing channel 132 together with the flow rates of the spheroidizing reagent and the blood sample can be set to expose the blood sample to the spherizing reagent for an appropriate time.

A sheath fluid (SH) pressure (such as sheath fluid (SH) pressure 36d of FIG. 3 ) may be provided to a sheath fluid reservoir (eg, sheath fluid reservoir 68 of FIG. 3 ). When pressure is applied, the sheath fluid is forced from the sheath fluid reservoir 68 into the sheath fluid channel 134 . In some embodiments, valve 136 and flow sensor 138 may be positioned in-line with sheath fluid channel 134 . Valve 136 can be controlled such that it opens when it is desired to push sheath fluid into the fluid circuit. The flow sensor 138 can measure the flow rate of the sheath fluid, and measure the flow rate of the sheath fluid flowing through the sheath fluid channel 134 . The flow rate provided by the flow sensor 138 may be used to help control the sheath fluid pressure (SH) provided to the removable cartridge 100 .

In this embodiment, the sheath fluid is provided to the confluence region 140 at the sheath fluid flow rate, and the spheroidized blood sample is provided to the confluence region 140 at the spheroidized blood sample flow rate. The flow rate of the spheroidized blood sample and the flow rate of the sheath fluid can be controlled by a pressure source/controller (such as the pressure source/controller 44 in FIG. 3 ).

The confluence region 140 may be configured in such a way that when the above two fluids flow through the confluence region 140, the sheath fluid flows around the spheroidized blood sample. In some cases, the sheath fluid flow rate is much higher than the spheroidized blood sample flow rate, which helps to promote nucleation in the downstream flow cell channel 142 . For example, in certain flow cytometry applications, junction region 140 may be configured to hydrodynamically concentrate and arrange spherified blood cells into single-row nuclei such that when red blood cells pass through optical cells in removable cartridge 100 When the window area 144 is removed, the analyzer can optically interrogate individual red blood cells. In some cases, fluid passing through flow cell channel 142 is directed to waste on the plate.

Turning now to the leukocyte measurement channel 104, the leukocyte solvent pressure (L) (eg, solvent pressure (L) 36b of FIG. 3) is provided to a solvent pool (eg, solvent pool 64 of FIG. 3). When pressure is applied, the solvent in the solvent pool 64 is forced into the solvent channel 154 .

In some embodiments, valve 156 and flow sensor 158 may be positioned in-line with solvent passage 154 . Valve 156 can be controlled such that it is opened when it is desired to push solvent into the fluid circuit. The flow sensor 158 may measure the flow rate of the solvent and measure the flow rate of the solvent flowing through the solvent channel 154 . The flow rate provided by flow sensor 158 may be used to help control the solvent pressure (L) provided to removable cartridge 100 by pressure source/controller 44 .

During normal functional operation of the removable cartridge 100, the solvent is provided to the junction region 160 at the solvent flow rate, and the blood sample is provided to the junction region 160 at the blood sample flow rate. Blood sample flow rate and solvent flow rate can be controlled by a pressure source/controller, such as pressure source/controller 44 of FIG. 3 .

The confluence region 160 may be configured to cause solvent to flow around the blood sample when the above two fluids flow through the confluence region 160 . In some cases, the solvent flow rate can be higher than the blood sample flow rate, which can help improve the flow characteristics in the downstream instant lysis channel 162, and in some cases, help form a thinner blood band completely and evenly surrounded by solvent . When the red blood cells flow through the immediate lysis channel 162 , this entrained flow can help the solvent dissolve the red blood cells evenly. Also, the length of the instant lysis channel 162, together with the flow rates of the solvent and blood sample, can be set to expose the blood sample to the solvent for an appropriate time.

A sheath fluid (SH) pressure (such as sheath fluid (SH) pressure 36d of FIG. 3 ) may be provided to a sheath fluid container (eg, sheath fluid container 68 of FIG. 3 ). When pressure is applied, the sheath fluid is forced from the sheath fluid reservoir 68 into the sheath fluid channel 164 . In some embodiments, valve 166 and flow sensor 168 may be positioned in-line with sheath fluid channel 164 . Valve 166 can be controlled such that it opens when it is desired to push sheath fluid into the fluid circuit. The flow sensor 168 can measure the flow rate of the sheath fluid and measure the flow rate of the sheath fluid flowing through the sheath fluid channel 164 . The flow rate provided by the flow sensor 168 may be used to help control the sheath fluid pressure (SH) provided to the removable cartridge 100 . In some cases, the flow rate of the sheath fluid flowing through the sheath fluid channel 164 is the same as the flow rate of the sheath fluid flowing through the sheath fluid channel 134 . However, in other cases, the flow rate of the sheath fluid flowing through the sheath fluid channel 164 may be different from the flow rate of the sheath fluid flowing through the sheath fluid channel 134 .

In this embodiment, the sheath fluid is provided to the junction region 170 at the sheath fluid flow rate, and the lysed blood sample is provided to the junction region 170 at the lysed blood sample flow rate. The flow rate of the lysed blood sample and the flow rate of the sheath fluid can be controlled by a pressure source/controller (eg, pressure source/controller 44 of FIG. 3 ).

The confluence region 170 may be configured in such a way that when the above two fluids flow through the confluence region 170, the sheath fluid flows around the dissolved blood sample. In some cases, the sheath fluid flow rate is much higher than the lysed blood sample flow rate, which can help promote nucleation in the downstream flow cell channel 172 . For example, in certain flow cytometry applications, junction region 170 may be configured to hydrodynamically concentrate and arrange leukocytes in a lysed blood sample into single-file nuclei such that when leukocytes pass through the optics of removable cartridge 100 When the window area 174 is opened, the analyzer can optically interrogate individual leukocytes. In some cases, fluid passing through the flow cell channel 172 is provided to a waste reservoir on the plate.

In some cases, absorption measurement channels can also be provided. In the illustrated embodiment, a portion of the lysed blood sample is provided to the absorption channel 180 . Valve 182 may be provided to selectively allow a portion of the lysed blood sample to pass through absorbent channel or region 184 . The analyzer may include a light source for illuminating the absorbing channel or region 184 and a detector for detecting light not absorbed by the lysed blood sample in the absorbing channel or region 184 . The analyzer can then determine the level of absorption from which a total absorption based hemoglobin measurement can be derived. In some cases, as shown in FIG. 8, an absorption channel 184 may be positioned downstream of the flow cell channel 172 if desired. In other cases, a whole blood sample may be provided directly (eg, from branch 116) to the absorption channel. In such cases, the absorption channel may include a mechanism to lyse red blood cells prior to performing the absorption measurement. Although the illustrated removable cartridge 100 is suitable for performing a complete blood count (CBC) analysis on a whole blood sample, it is believed that other removable cartridge configurations and analysis types may be used as desired.

Figure 5 is a schematic illustration of a plurality of storage pools that may be included in a removable cartridge according to the present invention. In the illustrated embodiment, a removable cartridge (eg, removable cartridge 100 of FIG. 4 ) may include a solvent reservoir 64, a push fluid reservoir 65, a spheroidizing reagent reservoir 66, a sheath fluid reservoir 68, a diluent reservoir 70, a staining agent pool 190 and waste pool 52. These examples are illustrative only, and we believe that more, fewer or different cells may be provided on or in the removable case.

The size of each well can be adjusted and the wells contain the appropriate amount of fluid and/or reagents to support the desired operation of the removable cartridge. Diluent reservoir 70 may contain a diluent fluid for diluting an input sample, such as a whole blood sample. In the embodiment of FIG. 4 , the spheroidizing reagent and/or solvent may perform the diluting function, therefore, a separate diluent reservoir 70 may not be necessary or even desirable. Likewise, in some embodiments, a stain pool, such as stain pool 190, may be required to stain the leukocyte channel to support leukocyte differentiation. We believe that the reagents and/or fluids stored in the cells may initially be in liquid or lyophilized form, depending on the specific application.

Figure 6 is a flow chart illustrating a method of analyzing a blood sample using a removable cartridge according to the present invention. In the method, a blood sample is first taken in step 200 . Next, a blood sample is provided to an anticoagulant-coated capillary in a removable cartridge. Then, the whole blood sample is separated, and the separated parts are respectively provided to a red blood cell and platelet (RBC/P) measurement channel 204 and a white blood cell (WBC) measurement channel 206 .

In the RBC/P measurement channel 204, as shown at 212, the erythrocytes are first spheroidized and then hydrodynamically concentrated, and the above-mentioned cells arranged in a single row are provided along the RBC/P flow cell channel 214 in the detachable box. red blood cells. As the red blood cells pass through the analysis region of the RBC/P flow cell channel 214, a light source 216, such as a vertical cavity surface emitting laser (VCSEL), shines light on each red blood cell. In some cases, an array of VCSELs is provided and only those VCSELs aligned with individual red blood cells are activated as they pass through the analysis region of RBC/P flow cytometry channel 214 . A portion of the incident light provided by the VCSEL is scattered, and detector 218 detects the scattered light. In some cases, detector 218 detects both forward angle scatter light (FALS) and small angle scatter light (SALS).

In some cases, a laser (or other) light source is focused (either as an elongated line source or as two separate point sources) in the RBC/P flow cell channel 214 . The RBCs and platelets in the RBC/P flow cell channel 214 pass through the above-mentioned focused light. High-quality light-harvesting optics can be used to form a sharp image of cells and focused illumination on an opaque screen comprising one, two or more parallel slits, preferably The longitudinal axis of the slot is set perpendicular to the direction of fluid flow in the RBC/P flow cell channel 214 . The distance between these slits is approximately the average cell spacing expected in RBC/P flow cytometry channels 21 . The opaque screen containing the slits may be placed in front of the one or more detectors 218 . As the image of the cell passes through the slit, it blocks light incident on the slit and causes a reduction in the signal on the detector 218, resulting in a pulse waveform whose width is proportional to the diameter of the cell. When two spaced apart slits are provided, the resulting two waveforms allow the calculation of the cell flow velocity, and thus the cell size. Using this technique, a high signal-to-noise ratio can be achieved, which allows easy event counting and identification of multiple cellular events. Pulse width and amplitude also allow one to distinguish certain cell types.

In some cases, images of the cells and the light source are formed on a double slit aperture placed in front of the detector 218 . The dual slit aperture provides a clearly defined geometric aperture and high signal-to-noise ratio for counting cells. As mentioned above, the signal from the slit allows one to make an accurate measurement of cell flow velocity, which in turn allows one to calculate the diameter of the cell.

In some cases, as shown at 220, a number of parameters may be measured during such an analysis, including sample flow rate (FR), length of measurement time (T), and sample dilution factor (DF). By monitoring the output of the detector and/or the corresponding scatter signature, the number of red blood cells (N _RB ), the number of platelets (N _Plt ), the diameter of individual cells (drbc) and the concentration of hemoglobin in individual cells can be measured.

From these parameters, as shown at 282, several red blood cell analysis parameters can be calculated, including red blood cell count (RBC=N _RB /(DF×FR×T)), platelet count (Plt=N _plt /(DF×FR×T)) , mean cell hemoglobin concentration (MCHC=<CHC>), mean cell volume (MCV=(π/6)×<drbc ³ >), mean cell hemoglobin content (MCH=(π/6)×<drbc ³ ×CHC> ), relative distribution width (RDW=[(π/6)×drbc ³ ]/standard deviation of MCV), cell-cell volume parameter (Hct=RBC×MCV) and/or hemoglobin concentration (Hb=MCHC×Hct).

In the WBC measurement channel 206 shown, red blood cells are first lysed as shown at 232, then they are hydrodynamically pooled and provided in a single file along the WBC flow cell channel 234 in the detachable cartridge. As each cell passes through the analysis region of the WBC flow cytometry channel 236, a light source 234, such as a vertical cavity surface emitting laser (VCSEL), illuminates each cell with light. In some cases, an array of VCSELs is provided, and as individual cells pass through the analysis region of WBC flow cytometry channel 234, only those VCSELs aligned with individual cells are activated. A portion of the incident light provided by the VCSEL is scattered, and detector 238 detects the scattered light. In some cases, detector 238 detects forward angle scatter light (FALS), small angle scatter light (SALS), and large angle scatter light (LASL). In some cases, as shown at 240, several parameters can be measured during this analysis, including on-axis cell volume, total WBC count, and WBC five (5) section differentiation.

The flow chart of Fig. 7 shows a method for obtaining a plurality of red blood cell parameters according to the present invention. In the method, at step 260 a blood sample is obtained. Next, as indicated at 264, the blood sample is diluted with a desired dilution factor (DF) and spheroidized. The above diluted and spheroidized blood cells are then hydrodynamically pooled and provided in a single file along the RBC/P flow cytometry channel in the removable cassette. When each cell passes through the analysis area of the RBC/P flow cytometry channel, a light source 216 (such as a vertical cavity surface emitting laser (VCSEL)) irradiates light on each cell. A portion of the incident light provided by the VCSEL is scattered, and detector 218 can be used to detect this scattered light. In some cases, detector 218 detects forward angle scatter light (FALS) and small angle scatter light (SALS) of individual cells. A processor or the like then maps the two independent scattering parameters (i.e. SALS and FALS) for each cell to a cell diameter parameter and a cell hemoglobin concentration parameter in the following manner:

{S _SALSi ，S _FALSi }->{drbc _i ，CHC _i }

As shown at 270 , if the intensity of the scattered S _SALSi plus S _FALSi is not greater than the predetermined detection threshold, then control returns to step 268 . However, if the intensity of the scattered S _SALSi plus S _FALSi is greater than the predetermined detection threshold, then control passes to step 272 . In step 272, it is determined whether the sum of S _SALSi and S _FALSi is greater than a predetermined platelet threshold. If S _SALSi plus S _FALSi is not greater than the predetermined platelet threshold, then particle "i" is determined to be a platelet and control passes to step 274 . In step 274 , the number of counted platelets (N _Plt ) is incremented by 1, and control is passed back to step 268 .

If the sum of S _SALSi plus S _FALSi is greater than the predetermined platelet threshold, the cell is a red blood cell and control passes to step 276 . Step 276 increments the counted number of red blood cells ( _NRBC ) by one and passes control to step 278 . In step 278, it is determined whether a predetermined measurement time has been reached. If not, then control passes back to step 268.

At step 278 , once the measurement time has been reached, control passes to step 280 . In step 280, a number of measured parameters are shown, including sample flow rate (FR), measurement time (T) length, sample dilution factor (DF), counted red blood cell number (N _RB ), counted platelet number ( N _plt ), diameter of individual cells ( _drbci ) and hemoglobin concentration (CHC _i ) of individual cells. According to these parameters, as shown in 282, several blood cell analysis parameters can be calculated, including red blood cell count (RBC=N _RBC /(DF×FR×T)), platelet count (Plt=N _plt /(DF×FR×T) ), mean cell hemoglobin concentration (MCHC=<CHC _i >), mean cell volume (MCV=(π/6)×<drbc _i ³ >), mean cell hemoglobin content (MCH=(π/6)×<drbc _i ³ ×CHC _i >), relative distribution width (RDW=[(π/6)×drbc _i ³ ]/standard deviation of MCV), hematocrit parameter (Hct=RBC×MCV) and/or hemoglobin concentration (Hb= MCHC×Hct), where the symbol <X _i > represents the average cell parameter calculated with all cells _Xi .

The flowchart of Fig. 8 shows another method of analyzing a blood sample according to the present invention. In the method, a blood sample is obtained and provided to a blood sample pool, as shown in step 300 . Next, the blood sample is provided to an anticoagulant-coated capillary in a removable cartridge and diluted. The whole blood sample is then separated, and the separated portions are provided to a red blood cell and platelet (RBC/P) measurement channel 304 and a white blood cell (WBC) measurement channel 340 , respectively.

In the RBC/P measurement channel 304, as shown at 306, red blood cells are first spheroidized, then they are hydrodynamically pooled and provided in a single row along the RBC/P flow cell channel 308 in the detachable cassette. cell. As each cell passes through the analysis region of the RBC/P flow cytometry channel 308, a light source 310 (eg, a vertical cavity surface emitting laser (VCSEL)) and its associated optics provide a focused light beam to each cell. In some cases, an array of VCSELs is provided such that only those VCSELs aligned with each cell are activated as each cell passes through the analysis region of the RBC/P flow cytometry channel 308 .

As individual cells/particles pass through the focused incident beam, a portion of the light is blocked, scattered or otherwise blocked, which can be detected by a detector (not shown). When two or more light sources are focused on different points spaced along the RBC/P flow cell channel 308, the front and/or back ends of individual cells can be detected. By measuring the time it takes the cells to move from one focal point to the next, the flow rate, and thus the cell velocity, can be determined. Using the determined cell velocity, the length of time the cell blocks, scatters, or otherwise obstructs the light beam can be correlated to cell size and/or cell volume.

In some embodiments, another light source 314 and associated optics may be provided by the analyzer. The associated optics of the light source 314 can collimate the light and measure off-axis scatter (eg, SALS and FALS scatter). Statistical red blood cell number ( _NRBC ) 316, statistical platelet number ( _NPlt ) 322, individual cell diameter (drbc), cell volume 318, and individual cell hemoglobin concentration ( CHC) 320. From these parameters, as described above, several blood cell analysis parameters can be calculated, including red blood cell count (RBC= _NRBC /(DF×FR×T)), platelet count (Plt=N _plt /(DF×FR×T)), Mean cell hemoglobin concentration (MCHC=<CHC _i >), mean cell volume (MCV=(π/6)×<drbc _i ³ >), mean cell hemoglobin content (MCH=(π/6)×<drbc _i ³ × CHC _i >), relative distribution width (RDW=[(π/6)×drbc _i ³ ]/standard deviation of MCV), hematocrit parameters (Hct=RBC×MCV) and/or hemoglobin concentration (Hb=MCHC× Hct), wherein, the symbol <X _i > represents the average cell parameter calculated with all cells _Xi .

In the WBC measurement channel 340 shown, red blood cells are lysed and dye injected as needed, as shown at 342 . The cells are then hydrodynamically concentrated and provided in a single file along the WBC flow cell channel 344 in the removable cassette. When each cell passes through the analysis region of the WBC flow cytometry channel 344, a light source 344, such as a vertical cavity surface emitting laser (VCSEL), shines light on each cell. In some cases, an array of VCSELs is provided such that as individual cells pass through the analysis region of WBC flow cytometry channel 344, only those VCSELs aligned with individual cells are activated.

As individual cells/particles pass through the focused incident beam, a portion of the light is blocked, scattered or otherwise blocked, which can be detected by a detector (not shown). When two or more light sources are focused on different points spaced along the WBC flow cell channel 344, the front and/or back ends of individual cells can be detected. By measuring the time it takes the cells to move from one focal point to the next, the flow rate, and thus the cell velocity, can be determined. Using the determined cell velocity, the length of time the cell blocks, scatters, or otherwise obstructs the light beam can be correlated to cell size and/or cell volume.

In some embodiments, a light source 350 and associated optics and/or polarizers may be provided. The associated optics of light source 350 can collimate the light and measure off-axis scatter (eg, SALS, FALS, and LALS scatter), as shown at 354 . As above, as shown at 352 , SALS, FALS and LALS scatter can be used to measure the number of white blood cells (N _WBC ) and assist in differentiating white blood cells, as shown at 356 . In some cases, one or more polarizers are provided to polarize the light provided by the source and the level of polarization extinction/rotation detected at the detector can be used to assist in leukocyte differentiation, however, this is not required in all implementations. Take this approach in the example.

In the illustrated embodiment, cells exiting the WBC flow cytometry channel 344 may be provided to a total absorption channel 360 . A light source 362 can illuminate cells in the absorption channel 360, and a detector 364 can detect light not absorbed by those resident cells. Thus, the absorption channel 360 can be used to measure the total absorption level of the resident cells. Rather, the level of absorption can provide a measure of the total or mean cellular hemoglobin concentration in the blood sample.

Figure 9 is a flowchart showing a method of operating a sample analyzer according to the invention. In the illustrated embodiment, a blood analysis cartridge (shown at 350) may be used and analyzed, a control cartridge may be used and analyzed to assist in verifying analyzer performance (shown at 370), and/or a calibration cartridge may be used and analyzed box to assist in calibrating the analyzer (shown at 380). The blood analysis cartridge can be loaded every time blood analysis is to be performed. Periodically (eg, once a day) the control box may be loaded into the analyzer to verify that the analyzer is producing accurate results. The analyzer can be recalibrated by loading a calibration cartridge into the analyzer less frequently (eg, every three months).

Each cartridge may contain all fluids and/or components necessary to perform the respective function. This way, the analyzer can be operated and/or maintained with very little training, while still achieving accurate results. Providing sample analyzers with removable cartridges and/or disposable cartridges that can be reliably used outside of a laboratory environment (requiring little or no specialized training) would help streamline the sample analysis process and reduce medical staff costs. cost and workload, and, for many patients (including those who require relatively frequent blood monitoring/analysis), increased convenience of sample analysis.

When a blood analysis cartridge is used, as shown at 350, a blood sample is collected and loaded into the blood analysis cartridge, as shown at 352 and 354. The blood sample is drawn into the blood analysis cartridge by capillary action or manual pumping, as required. Then, the blood analysis cartridge can be loaded into the analysis instrument. In the illustrated embodiment, after performing the above steps, the analyzer can self-align the blood analysis cartridge and the corresponding components of the analyzer (such as light source/light detector, etc.), as shown at 356 . Next, one or more buttons may be pressed to initiate the blood analysis process. Rather than pressing a button or the like, in some cases the mere step of loading the cartridge into the analyzer can cause the analyzer to initiate the alignment and blood analysis process described above.

Once the analyzer is turned on, the analyzer can perform several tests. For example, an analyzer can close all valves on a blood analysis card and apply pressure to various fluid ports on the card. The analyzer can then measure the flow rate through one or more flow sensors on the card. Flow should be zero since all valves are closed. However, the analyzer can recalibrate the flow sensor to zero flow if the flow sensor indicates a non-zero flow rate. This can help improve the accuracy of the flow sensor measurements. Alternatively or additionally, the analyzer can check for blood clotting in the removable cartridge by measuring the flow rate of the blood sample (e.g. using a flow sensor) and the applied pressure, determining if the flow rate appears to be too low relative to the applied pressure Has solidified. If blood clotting is detected, the analyzer may display a message indicating that the measurement has failed.

The analyzer can then implement the blood analysis cartridge timing protocol. The blood analysis cartridge timing protocol may be similar to that shown and described in US Patent Application Serial No. 10/932,662, assigned to the assignee of the present invention and incorporated herein by reference. The exact timing protocol for the blood analysis cartridge depends on the specific design of the blood analysis cartridge. The analyzer can also verify the presence of steady nuclear flow in any flow cell channel on the blood analysis cartridge and, if there is such a nuclear flow, identify its location.

The cartridge can then lyse the red blood cells from the portion of the blood sample that will be used for white blood cell measurements and spherize the red blood cells from the portion of the blood sample that will be used for red blood cell measurements to form nuclei in any flow cytometry channel on the cartridge. stream, and/or perform any other desired functionality. The analyzer can provide light to a selected area of the blood analysis cartridge (eg, any flow cytometry channel) and detect light passing through the selected area.

Accordingly, the analyzer can count and classify the particles (such as white blood cells, red blood cells, platelets, etc.) in the sample, and then display, print, make a sound or indicate the blood analysis results to the user in other ways. In certain embodiments, the analyzer displays or prints quantitative results (eg, within and/or outside predefined ranges), which eliminates the need for further calculations or interpretation by the user. Finally, the blood analysis cartridge can be removed from the analyzer and discarded.

As shown at 370, a control box may be used when control operations are to be performed. In some cases, the control operation may be performed periodically, such as once a day or once a week. A control box may contain a control sample with known characteristics. Therefore, when the analyzer performs the analysis on the control sample, a known result should be obtained. In the method shown, a control box is loaded into the analyzer, as shown at 372 . Next, as shown at 374 , the analyzer is started, and the analyzer performs the analysis and displays the results, as shown at 376 . In certain embodiments, the analyzer displays or prints quantitative results (eg, within and/or outside predefined ranges) so that no further calculations or interpretations are required by the user. Finally, remove the control box from the analyzer and discard it. If the results of the control run are outside predefined ranges, a calibration run, such as calibration run 380 , may need to be performed.

As indicated at 380, a calibration cartridge may be used when a calibration run is to be performed. In some cases, calibration runs may be performed periodically, such as once a month or otherwise as desired. Calibration boxes may contain calibration samples with known characteristics. Thus, when the analyzer performs analysis on a calibration sample, known results should be obtained. In the method shown, a calibration cartridge is loaded into the analyzer, as shown at 382 . Next, as shown at 384, the analyzer is started and several results are obtained. By comparing the results obtained during a calibration run with the expected results, the analyzer can automatically adjust one or more calibration factors in memory to recalibrate the analyzer so that subsequent runs will produce the expected or expected results. The desired result, as shown at 386.

Figure 10 is a flowchart illustrating another method of operating a sample analyzer in accordance with the present invention. The method is indicated generally at 400 and begins at step 402 . Control passes to step 404 where a blood sample is provided to the disposable fluid cartridge. Control then passes to step 406 where the disposable fluid cartridge is inserted into the blood sample analyzer. Control then passes to step 408 . In step 408, the blood sample analyzer is started, and then in step 410, blood analysis results are obtained from the blood sample analyzer without any further intervention by a user of the blood sample analyzer. Control then passes to step 412 where the above method exits.

Figure 11 is a flowchart illustrating another method of operating a sample analyzer in accordance with the present invention. The method is indicated generally at 500 and begins at step 502 . Control passes to step 504 where a blood sample is provided to the disposable fluid cartridge. Control passes to step 506 where the disposable fluid cartridge is inserted into the blood sample analyzer. Control then passes to step 508 . In step 508, the blood sample analyzer is started, and then in step 510, blood analysis results are obtained from the blood sample analyzer without any further intervention by a user of the blood sample analyzer. Control then passes to step 512 . In step 512, it is determined whether the blood analysis result is within a predefined range. As noted above, in some embodiments, the analyzer can display or print quantitative results (eg, within and/or outside predefined ranges), which eliminates the need for further calculations or interpretation by the user. Control is then passed to step 514 where the above method exits.

Figure 12 is a flowchart illustrating another method of operating a sample analyzer in accordance with the present invention. The method is indicated generally at 600 and begins at step 602 . In this embodiment, a blood analysis cartridge may be used and analyzed (shown at 604), a control cartridge may be used and analyzed to assist in verifying analyzer performance (shown at 620), and/or a calibration cartridge may be used and analyzed , to assist in calibrating the analyzer (shown as 640). The blood analysis cartridge can be loaded every time blood analysis is to be performed. Periodically (eg, once a day) the control box may be loaded into the analyzer to verify that the analyzer is producing accurate results. The analyzer can be recalibrated by inserting a calibration cartridge into the analyzer less frequently (eg, every three months) or as needed.

Each type of cartridge may contain all necessary fluids and/or components to perform the respective function. As a result, very little training is required to operate and/or maintain the analyzer and still achieve accurate results. Providing sample analyzers with removable and/or disposable cartridges that can be reliably used outside of a laboratory setting (requiring little or no specialized training) can help streamline the sample analysis process and reduce costs for medical staff and workload, as well as increased convenience of sample analysis for many patients, including those requiring relatively frequent blood monitoring/analysis.

In the method shown in FIG. 12 , control passes to step 604 when the cartridge is to be used. In step 606, a blood sample is provided to a disposable fluid cartridge. Control then passes to step 608 where the disposable fluid cartridge is inserted into the blood sample analyzer. Control then passes to step 610 . In step 610, the blood sample analyzer is started, and in step 612, blood analysis results are obtained from the blood sample analyzer.

Control passes to step 620 when the control box is to be used. Step 620 passes control to step 622 where the control cartridge is inserted into the blood sample analyzer. Control then passes to step 624 . In step 624, the blood sample analyzer is started, and then, in step 626, blood analysis results are acquired using the control fluid cartridge. Control then passes to step 628 . In step 628, it is judged whether the control analysis result is within the expected control range. If the control analysis results are not within the expected range, the results obtained by the blood analysis cartridge should not be trusted. In some cases it is possible to run the calibration box to recalibrate the sample analyzer and then verify the sample analyzer operation/calibration via another control box.

Control passes to step 640 when a calibration cartridge is to be used. Then, go to step 642 . In step 642, the calibration cartridge is inserted into the blood sample analyzer. Control then passes to step 644 . In step 644, the blood sample analyzer is turned on, and then, in step 646, the calibration fluid cartridge is used to obtain calibration analysis results. Control then passes to step 648. In step 648, the analyzer is adjusted if necessary based on the calibration analysis results.

Claims (54)

1. A disposable analysis cartridge capable of performing at least part of a blood sample measurement, comprising: two or more flow cytometry channels, each channel adapted to hydrodynamically focus the sample and support light scattering measurements; and A fluid directing device for directing sample fluid flow to at least one of the two or more flow cytometry channels. 2. The disposable analysis box as claimed in claim 1, further comprising: One or more absorption-based measurement channels. 3. The disposable analysis box as claimed in claim 1, further comprising: one or more channels in fluid communication with the fluid directing device; One or more flow sensors for measuring flow velocity in at least selected ones of the one or more channels. 4. The disposable analysis cartridge as claimed in claim 3, further comprising: One or more pressure sensors for measuring pressure in at least selected ones of the one or more channels. 5. The disposable analysis cartridge as claimed in claim 4, further comprising: A controller for determining whether the fluid in the at least selected ones of the one or more channels has a viscosity outside a predetermined threshold. 6. The disposable analysis cartridge as claimed in claim 1, further comprising: One or more storage pools, one for each reagent. 7. The disposable assay cartridge of claim 6, wherein said reagent is selected from the group consisting of isotonic solution (dilute), hypotonic solution (dissolve), nodulizing agent, buffering agent, hemolyzing agent , antibodies, cytochemical stains, fluorescent stains. 8. The disposable assay cartridge of claim 7, wherein the reagents are in liquid form. 9. The disposable assay cartridge of claim 7, wherein the reagents are in lyophilized form. 10. The disposable assay cartridge of claim 1, further comprising a whole blood collection device on board. 11. The disposable assay cartridge of claim 10, wherein the whole blood collection device is a lancet. 12. The disposable assay cartridge of claim 1, further comprising a whole blood storage circuit on board. 13. The disposable assay cartridge of claim 12, wherein at least a portion of the whole blood storage circuit on the plate is coated with an anticoagulant. 14. The disposable assay cartridge of claim 13, wherein the anticoagulant is ethylenediaminetetraacetic acid. 15. The disposable analysis cartridge of claim 1, wherein a first channel of the one or more flow cytometry channels is adapted to support measurement of one or more white blood cell parameters, and the one or more flow cytometry channels A second channel of the or more flow cytometry channels is adapted to support measurement of one or more red blood cell parameters. 16. The disposable analysis cartridge of claim 15, wherein at least one of the one or more white blood cell parameters is related to a total white blood cell count. 17. The disposable assay cartridge of claim 15, wherein at least one of the one or more leukocyte parameters is related to leukocyte differentiation. 18. The disposable assay cartridge of claim 15, wherein at least one of the one or more white blood cell parameters is related to cell volume. 19. The disposable analysis cartridge of claim 15, wherein at least one of the one or more red blood cell parameters is related to red blood cell count. 20. The disposable analysis cartridge of claim 15, wherein at least one of the one or more red blood cell parameters is related to platelet count. 21. The disposable assay cartridge of claim 15, wherein at least one of the one or more red blood cell parameters is related to cell diameter. 22. The disposable assay cartridge of claim 15, wherein at least one of the one or more red blood cell parameters is related to hemoglobin concentration. 23. The disposable analysis cartridge of claim 15, further comprising one or more storage pools, each storage pool for storing a reagent, wherein at least one of the reagents is connected to the The sample fluid of the first channel of two or more flow cytometry analysis channels is mixed. 24. The disposable analysis cartridge of claim 23, wherein the reagent mixed with the sample fluid introduced to the first of the two or more flow cytometry channels Is red blood cell hemolysis agent. 25. The disposable assay cartridge of claim 24, wherein at least one of said reagents is associated with said second channel of said two or more flow cytometry analysis channels. mixed with the sample fluid. 26. The disposable analysis cartridge of claim 25, wherein the reagent mixed with the sample fluid introduced to the second of the two or more flow cytometry channels It is a red blood cell spheroidizing agent. 27. The disposable assay cartridge of claim 1, further comprising an absorption channel adapted to support light absorption measurements. 28. The disposable analysis cartridge of claim 1, further comprising an extinction channel adapted to support extinction measurements. 29. A method for analyzing a blood sample comprising: providing a portion of the blood sample to a first measurement channel in a disposable analysis cartridge; providing a portion of the blood sample to a second measurement channel in the disposable analysis cartridge; lysing red blood cells from the blood sample after providing the sample to the first measurement channel in the disposable cartridge; providing the lysed sample of red blood cells to a first flow cytometric analysis channel, wherein the first flow cytometric analysis channel is adapted to hydrodynamically focus the lysed sample of red blood cells; and Directing a light beam to the red blood cells in the first flow cytometry channel yields a lysed sample and detects light scattered energy at two or more detector angles. 30. The method of claim 29, further comprising the step of determining one or more white blood cell parameters from said detected light scatter energy from said first flow cytometry channel. 31. The method of claim 29, further comprising the step of staining one or more leukocytes in the blood sample after providing the sample to the first measurement channel in the disposable cartridge . 32. The method of claim 29, further comprising differentially lysing one or more of the blood samples after providing the samples to the first measurement channel in the disposable analysis cartridge Leukocyte steps. 33. The method of claim 29, further comprising the step of: spheroidizing the red blood cells in the blood sample after providing the sample to the second measurement channel in the disposable analysis cartridge; providing the spheroidized sample of red blood cells to a second flow cytometric analysis channel, wherein the second flow cytometric analysis channel is adapted to hydrodynamically focus the spheroidized sample of red blood cells; and Directing a beam of light to the red blood cells in the second flow cytometry channel yields a spheroidized sample and detects light scatter energy at two or more detector angles. 34. The method of claim 29, further comprising the step of detecting extinction using one or more detectors. 35. The method of claim 33, wherein the spheroidizing of red blood cells is done extemporaneously on a disposable cartridge. 36. The method of claim 35, wherein said spheroidizing is performed by mixing a spherifying agent with said red blood cells. 37. The method of claim 36, wherein the exposure time of the red blood cells to the spheroidizing agent is controlled within a predetermined time range. 38. The method of claim 33, further comprising the step of determining one or more red blood cell parameters from said detected light scatter energy from said second flow cytometry channel. 39. The method of claim 33, further comprising the step of providing two or more wavelengths of light to the sample in the second flow cytometry channel. 40. The method of claim 33, further comprising the step of polarizing the light beam prior to providing the light beam to the sample in the second flow cytometry channel. 41. The method of claim 40, further comprising the step of detecting polarization extinction using one or more detectors. 42. The method of claim 33, further comprising the step of determining two or more red blood cell parameters from said detected light scatter energy from said second flow cytometry channel. 43. The method of claim 33, further comprising the step of: A light beam is provided to the sample in the second flow cytometry channel, and an absorbance value corresponding to light absorbed by the sample in the second flow cytometry channel is detected. 44. The method of claim 33, wherein the one or more detector angles comprises an on-axis detector angle. 45. The method of claim 33, further comprising the step of determining a refractive index measurement of at least one red blood cell. 46. The method of claim 33, further comprising the step of determining a diameter measurement of at least one red blood cell. 47. The method of claim 33, further comprising the step of determining a sample volume of said sample passing through said second flow cytometric analysis channel. 48. A method for analyzing a blood sample comprising: providing a portion of the blood sample to a first measurement channel in a disposable analysis cartridge; providing a portion of the blood sample to a second measurement channel in the disposable analysis cartridge; spheroidizing red blood cells in the blood sample after providing the sample to the second measurement channel in the disposable analysis cartridge; providing the spheroidized sample of red blood cells to a first flow cytometric analysis channel, wherein the first flow cytometric analysis channel is adapted to hydrodynamically focus the spheroidized sample of red blood cells; and Introducing a beam of light to the red blood cells in the first flow cytometry channel yields a spheroidized sample and detecting light scatter energy at one or more detector angles. 49. A disposable analysis cartridge capable of performing at least a portion of a blood sample measurement, comprising: two or more flow cytometry channels, at least a first of said flow cytometry channels is adapted to hydrodynamically concentrate a sample, and is adapted to analyze channels to support electrical measurements of said samples; and Fluid directing means for directing fluid to at least said first of said flow cytometry channels. 50. A disposable analysis cartridge capable of performing at least a portion of a blood sample measurement, comprising: two or more flow cytometry channels, at least a first of said flow cytometry channels is adapted to hydrodynamically concentrate a sample, and is adapted to analyze channel supports the measurement of fluorescent labels on the sample; and Fluid directing means for directing fluid to at least said first of said flow cytometry channels. 51. A disposable analysis cartridge capable of performing at least a portion of a blood sample measurement, comprising: two or more flow cytometry channels, at least a first of said flow cytometry channels is adapted to hydrodynamically concentrate a sample, and is adapted to analyze channels to support light absorption and/or extinction measurements of the sample; and Fluid directing means for directing fluid to at least said first of said flow cytometry channels. 52. The disposable assay cartridge of claim 51, further comprising an absorption channel adapted to support light absorption measurements, and wherein the fluid directing means is adapted to direct fluid to the absorption channel. 53. The disposable analysis cartridge of claim 51, further comprising an extinction channel adapted to support extinction measurements, and wherein the fluid directing means is adapted to direct fluid to the extinction channel. 54. A disposable analysis cartridge capable of performing at least a portion of a blood sample measurement, comprising: two or more reagent storage pools, a first storage pool for storing a hemolytic agent and a second storage pool for storing a nodulizing agent; a first flow sensor in fluid communication with the first one of the reagent reservoirs; a second flow sensor in fluid communication with the second one of the reagent reservoirs; an immediate lysis channel in fluid communication with said first one of said reagent reservoirs; an instant spheroidization channel in fluid communication with the second one of the reagent reservoirs; a first flow cytometry channel for hydrodynamically focusing a sample and for supporting light scattering measurements, said first flow cytometry channel being in fluid communication with said immediate lysis channel; and A second flow cytometry channel for hydrodynamically focusing the sample and for supporting light scattering measurements is in fluid communication with the instant spherification channel.

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